C Z E C H M Y C O L . 48 (1 ) , 1995

Fungi in biotechnology. Past, present, future

H A N S P E T E R M O L I T O R I S

Botanischer Institut, Abteilung Pilzphysiologie, Universitat Regensburg, Postfach, D-93040 Regensburg, BRD

Molitoris H.P. (1995): Fungi in biotechnology. Past, present, future.- Czech Mycol. 48: 53-65

From multiple uses of fungi in early historic times, "early biotechnological processes" devel­oped. Fungi were used here already e.g. for production of cheese, bread, wine, beer and other foodstuff.

Based on these processes, fungi today have become one of the most important group of organisms in modern technology, where food, fodder and various metabolites such as antibiotics, enzymes, steroids etc. are produced on an industrial scale.

An equally important role of fungi in biotechnology is also to be expected for the future where - in addition to existing uses - fungi will be increasingly used employing modern methods such as genetic engineering and will also new applications as in pollution control, biological control of pests, microbial leaching and even biotechnology.

Key words: Fungi, biotechnology, past, present, future

Molitoris, H.P. (1995): Houby v biotechnologiich. Minulost, pfitomnost, budoucnost.-Czech Mycol. 48: 53-65

Z cetnych pouziti hub v rannych historickych dobach se vyvinuly „ranne biotechnologicke metody". Houby byly pfi nich uz pouzivany pfi vyrobe syru, chleba, vina, piva a dalsich potravin. Houby se dnes staly, na zaklade techto metod, jednou z nejdulezitejsich skupin organismu v modernich biotechnologiich, kdy potraviny, krmiva a ruzne metabolity jako antibiotika, enzymy, steroidy atd. se vyrabeji na prurnyslovem zaklade. Rovnocenna role hub v biotechnologiich je ocekavana i v budoucnu, kdy - krome rady dosavadnich pouziti - budou houby vyuzivany za ucasti modernich metod, jako je geneticke inzenyrstvi a naleznou vyuziti pfi zvladani znecisteni prostfedi, biologickem potlacovani skudcu, mikrobialnim vyluhovani a mozna i v biotechnologiich v kosmickem prostoru.

I N T R O D U C T I O N A N D DEFINITION

Fungi are becoming more and more important i n our life. Therefore, as a start for the symposium on F I L A M E N T O U S F U N G I I N B I O T E C H N O L O G Y a short survey of the role of fungi i n biotechnology in the past, in the present and in the foreseeable future shall be presented (see also Molitoris 1989, 1990a).

Biotechnology is a relatively new term meaning "the use of organisms for the production of biomass or metabolites on an industrial scale". B I O T E C H N O L O G Y involves and interconnects the fields of B I O L O G Y (the organisms involved), E C O ­N O M I C S (costs), K I N E T I C S (physical/chemical influences on biotechnological processes) and P R O C E S S M A N A G E M E N T (see Lafferty 1981).

F U N G I A N D M A N

In their relation to man, fungi may be considered as F O E or as F R I E N D (Table 1).

Tab. 1 Fungi and Man

F O E F R I E N D Spoilage Mineralization Pathogen Mykorriza Allergen Food and Fodder Toxin production Medicine

Religious/mystical ceremonies Science and Research Biotechnology Biological control Pollution control

Examples for the negative action of fungi are their activities i n causing S P O I L A G E of natural substrates (dry-rot of wood in buildings by Serpula lacrymans) and man-made substrates (plastics by Penicillium simplicissimum), their action as P A T H O G E N S F O R P L A N T S (corn-smut of maize, Ustilago maydis), for animals and man in the latter case not only as A L L E R G E N but also causing often severe diseases (blastomycosis by Blastomyces dermatitis) or I N T O X I C A T I O N S (poisoning by consumption of Amanita phalloides poisoning).

In many respects, however, fungi have important and positive values i n nature and for man: They are part of the microbiota in soil causing M I N E R A L I Z A T I O N by degradation of organic material and as M Y C O R R H I Z A they are important symbionts for many higher plants, especially trees. Coming to the topic of this review, they are used in the production of F O O D and F O D D E R ; since ancient times they are used i n M E D I C I N E (Molitoris 1978, 1994) and more recently they have became important tools in S C I E N C E and R E S E A R C H and are used increasingly to fight increasing problems of o ur modern society i n the B I O L O G I C A L C O N T R O L O F P A T H O G E N S and in P O L L U T I O N C O N T R O L .

F U N G I IN B I O T E C H N O L O G Y IN T H E PAST In the history of mankind, fungi have been used in various ways for a variety of

purposes (Table 2).

Tab. 2 Fungi in biotechnology in the past

E A R L Y G E N E R A L U S E S O F F U N G I

E A R L Y B I O T E C H N O L O G I C A L P R O C E S S E S

Food Poison Medicine Mystical/religious ceremonies

Cheese Bread Beer Wine Cultivated edible mushrooms

Early general uses of fungi

Food Since earliest times, fungi have been widely used as F O O D as symbolized i n the

medieval fresco of the chapel of Plaincorault in France (1291 a . C ) where A d a m and Eve in the famous temptation scene are seen eating from a "fungus-tree" (Wasson 1968).

Poison Fungi were important also as P O I S O N , either consumed accidentally or admin­

istered purposely like in the alleged poisoning of the Roman emperor Claudius by Agrippina (Ramsbottom 1972). They were also used for causing artificial abortion by the "wise women" in the middle ages which gave for this purpose the alkaloid-containing sclerotia of ergot (Claviceps purpurea), a fungal parasite of grasses.

Medicine Ergot by the way, was used also as M E D I C I N E already in the middle ages in

gynaecology and other areas, as also many other fungi have been used as medicinal plants (Molitoris 1978, 1994).

Mystical/religious ceremonies Some fungi, i n particular hallucinogenic ones, such as Psilocybe cubensins or

Amanita muscaria have been used by many ethnic groups in mystical, long-forgotten religious or shamanic ceremonies, which often date back thousands of years as documented by the so-called "fungus stones" from Guatemala in Middle America (Heim, 1963).

"Early biotechnological processes" involving fungi The use of fungi by early man led i n some cases o processes which could well be

termed "early biotechnological" processes by procedure importance, amount and value of the products.

Cheese One of the earliest examples would be the production of milk products such

as C H E E S E . When man turned from gatherer and hunter to be nomad and later farmer, herds of domesticated cattle gave meat, milk and other goods. Milk is one of the most nourishing but also perishable natural goods, which by action of microorganisms either can be destroyed but also can become a stable, nutritious and delicious food like cheese. One of the earliest documents for the use of milk is an almost 4000 year-old relief inthe sumeric town of Ur showing the whole procedure from getting the milk until making butter and/or chease.

Bread Another early example would be the preparation of B R E A D from ground cereals

where yeasts under appropriate conditions (water, temperature) are responsible for the leavening of the dough by their carbon dioxide production. Early bread-making has been shown in an old Egyptian relief dating back about 4500 years.

Already in ancient times B E E R and WINE, two alcoholic beverages, were produced in large amounts and played an important role as recreational drinks, but also as necessary food. Here fungi (yeasts) convert the sugar of cereals and grapes into carbon dioxide and alcohol.

Wine For making WINE particularly the Greeks, Jews and Romans were known

in ancient times. This is often documented in sculptures, paintings and written documents as in the Holy Bible (the miracle of the wedding of Kanaan).

Beer Also the history of beer-making goes back a very long time as is illustrated by

the famous relief from ancient Babylon, showing two persons drinking beer with straws from an amphora. The explanation for this unusual way of drinking beer is of course that filtration of beer in ancient times was an unknown technique. Therefore the fresh beer was cleared first by sedimentation of the particles and then the supernatant beer was drunk with straws. It might be also interesting to know that the old Babylonians knew already about 20 different kinds of beer.

B E E R and WINE became more and more important as food and even as medicine (every Roman soldier had to drink daily 2 litters of wine for his health - happy times!). No wonder that some of the oldest food laws were concerned with these liquids. The German "purity law for beer" of 1516 exactly regulated ingredients, production and marketing of beer and punished those not obeying it. This law is still in effect in Germany.

Cultivated edible mushrooms Cultivation of edible mushrooms most probably started more than thousand

years ago in the Far East with mushrooms such as Lentinus edodes ("Shii-take").

In middle Europe the first reports are from the Middle ages about cultivation of the edible mushroom Agaricus bisporus i n caves i n France.

F U N G I IN B I O T E C H N O L O G Y IN T H E P R E S E N T

From the impressive list given i n table 3, just a few items wi l l be discussed.

Tab. 3 Fungi in biotechnology in the present

F O O D P R O D U C T I O N O F F O O D A N D F O D D E R

Mushrooms Fungal biomass

Edible Asiatic fermentation products Alcoholic beverages Milk products

M E T A B O L I C P R O D U C T S Alcohol Organic acids Vitamins Steroids Amino Acids Hormones Antibiotics Nucleic acids Ergot alkaloid Enzymes

R E S E A R C H O T H E R Genetics Metabolism

Biological control Pollution control Recycling

Food

Fungi as food presently play an important role in our economy by amount and value.

Edible mushrooms Edible fungi for human consumption are cultivated nowadays in highly mecha­

nized, automatized and even computerized enterprises world-wide. This production is constantly increasing by rising demand, more efficient procedures, but w i t h decreasing prices. Advantages of the cult ivation of edible mushrooms are cheap substrates - even waste products such as straw - no need to use arable land, excellent flavour, high v i tamin and mineral content, and i n some cases even positive health effects such as in Lentinus edodes.

World-wide, the production of cultivated edible mushrooms reaches now more than 4 mi l l ion tons per year, w i th the cultivated mushroom, Agaricus bisporus, leading, followed by the oyster mushroom, Pleurotus ostreatus, the Shii-take mushroom, Lentinus edodes, and others as shown in table 4.

Tab. 4 World production of cultivated mushrooms in 1991 (from Chang 1993)

Species (common name) Production (tons x 1000)

Agaricus bisporus and other spp. (cultivated mushroom) 1,590 Pleurotus ostreatus and other spp. (oyster mushroom) 917 Lentinus edodes (Shii-take) 526 Auricularia auricula-judae (Jew's ear mushroom) 465 Volvariella volvacea (straw mushroom, Chinese mushroom) 253 Others 531 Total 3,742

Biomass In contrast to this, fungal biomass production from submerged cultivation on

originally cheap hydrocarbons (single cell protein = S C P ' s ) , which was intended for fighting hunger i n third-world-countries, d id not fulfil the expectations because of rising costs of the substrates and for food preferences in those countries for which the product originally was meant for.

The only exception is Q U O R N , a B r i t i s h fermentation product from the Deuteromycete Fusarium graminearum which fulfils strict foodstuff requirements, has good nutr i t ional values and seems to be produced at compatible prices.

Production of food and fodder

Presently about 25% of our food is already produced by biotechnological processes and fungi play an important part in i t .

Alcoholic beverages In the western world W I N E and B E E R are leading i n importance and also

this country is famous for these products. So one of the great typical beers, the "Pilsener" w i t h its typical taste, originates from the Czech town of Pi lsen, where it was originally brewed.

The production of W I N E , the other major alcoholic drink, is s t i l l increasing. B y new and improved methods and new breeds of grapes, new areas such as Cal i fornia and Chi le i n Amer ica , South Afr i ca and Austra l ia , in recent decades started to grow and sell quality wine w i t h increasing success.

Milk products C H E E S E is s t i l l one of the most important fungal products from milk. A number

of these cheeses are quite typical for certain areas and are even named for them, e.g. "Roquefort" (caves of Roquefort, Prance), "Edamer" (town i n Hol land) , "Emmentaler" (valley in Switzerland), "Danish B lue " or "Bavar ian Blue" and last not least the "Camembert" from witch the producing fungus, Penicillium

camemberti, got its name. More recently, also other fungal products from milk, such as Kef ir , Yoghurt and others, gain more and more importance.

Metabolic products

One of the main areas where fungi are used today i n biotechnology, is in the production of certain metabolites for various purposes as given in table 3.

Alcohol Alcohol is s t i l l a fungal product of major importance. It has changed, however,

insofar as it is not only used in beverages, but is used increasingly as a component of medicines and for preparative and synthetic processes in industry. More recently, however, the amount of alcohol produced from prokaryotes, is increasing.

Steroids Steroids became very important since the advent of the anti-baby p i l l and also

for other medical purposes. Fungi such as several Penicillium as Aspergillus strains by virtue of their abil ity to catalyse very specific one- or few-step transformations in steroids became essential in the production of these drugs.

Antibiotics Since Alexander F leming in 1928 discovered the production of the first antibiotic,

Penic i l l in , from the imperfect fungus Penicillium notatum, everyone knows about the importance of these metabolites i n medicine. About 6000 antibiotics are known today, at least by their formula, but only about 400 of them, for various reasons, are used i n human medicine. Fungi produce about a quarter of them. Constantly new antibiotics are looked for and found, a necessity by the fact that pathogenic microorganisms constantly acquire resistance against the known and administered antibiotics.

Enzymes Quite a number of fungal enzymes are produced commercially and have become

indispensable tools i n industry and research (Table 5). A s the table shows, they are used for a wide variety of applications from biochemical analysis, over medical diagnosis to food processing.

Organic acids, amino acids, nucleic acids A m o n g the organic acids of fungal origin, citric acid is leading wi th an annual

production of over 600.000 tons. It is mainly used in the food and beverage industry. But also amino acids and nucleic acids are commercially produced from fungi and find wide application.

Ergot alkaloids Fungal alkaloids, mainly originating from ergot, Claviceps purpurea, have been

mentioned earlier mainly as poison, an abort ivum and occasionally as medicinal drug. The latter use nowadays is their main importance. These alkaloids are

produced now in increasing amount by saprophytic culture or even by biosynthesis using certain - even genetically engineered - strains. These alkaloids find now wide medicinal application e.g. i n gynaecology, against migraine and i n the treatment of heart and circulatory diseases.

Tab. 5 Important industrial enzymes from fungi (Molitoris 1991)

Enzyme Use O X I D O R E D U C T A S E S ( E C 1.) Glucose oxidase Catalase

Taste intensifier Removal of hydrogenperoxide

H Y D R O L A S E S ( E C 3.) Esterases (Ec 3.1) Lipase Aroma development, aid for extraction Ribonuclease Taste intensifier

Glucosidas ( E C 3.2) Q-Amylase Baking and brewing industry /3-Amylase Starch hydrolysis Dextranase Cosmetic industry (tooth paste) Glucoamylase Starch hydrolysis, brewing industry Hemiceilulase Food industry Invertase Production and processing of sweets Lactase Milk- and baking industry Melibiase Sugar production from sugar beets Naringinase Beverage industry (removal of bitter taste) Pectinase Beverage industry (fruit juices) Xylanase Food industry Cellulase Food-, cellulose-, paper-industry, SCP production

Peptide hydrolases ( E C 3.4) Protease Food industry, medicine (peptic preparation, digestion) Rennin Milk industry (cheese production)

Research

Genetics

Because fungi possess a relatively low level of organisation and are easy to handle i n the laboratory, fungi represent increasingly important tools i n modern research, especially i n genetics. The different types of life cycles, the different ways of genetic inheritance and e.g. the possibility to identify by tetratede analysis in certain

ascomycetes the stepwise production of the meiosis products, are advantages of this group of microorganisms. Successes in this area of research are documented e.g. by the first Nobel Prise given for fungal research in 1958 for physiology and medicine to B E A D L E and T A T U M for their work with the red bread mould Neurospora crassa.

Metabolism Fungal metabolites are indispensable substances in modern research. Fungal

antibiotics e.g. already mentioned as highly efficient medicaments, are used also in research. The antibiotics Penic i l l in , whose action against many pathogenic bacteria is based on its effect on the bacterial cell-wall synthesis, is used to analyse and identify specific steps in this important morphogenetic process.

Other

Biological control of pathogens To reduce the need for chemicals in control of pests and pathogens, recently

much work has been done to find biological control measures. These methods would reduce the need for chemicals, which are expensive, relatively unspecific and also cause environmental problems because of their toxicity and recalcitrance. Again , in this quite modern aspect of biotechnology, fungi are involved, particularly because they show a high specificity of action (Burge 1988).

Nematodes, a deadly pest for mushroom culture, can efficiently be controlled by certain fungal preparations (Stirl ing 1988), such as from Arthrobotrys robusta ("Royal 300").

Some of the enthomopathogenic fungi, fungi pathogenic against insects, can be used for control of insect pest, such as "Myco ta l " and "Vertalec" , preparations from Verticilium lecanii, which is used i n the green houses industry (Quinlan 1988).

A n d finally, fungi may be used to fight fungal diseases as the Deuteromycete Trichoderma can be used against a number of pathogenic fungi, in particular if used as "integrated control" in connection with other control measures (Papavizas and Lewis 1988).

Pollution control As already indicated above, biological control measures using fungi in turn help

in reducing the need for chemicals i n pest and pathogen control. They therefore are one new and important aspects in the involvement of fungi i n the ever more necessary measures for the protection of our environment.

Recycling Recycling in other word, making use of materials which otherwise would

contribute to pol lution or are at least useless, can consider as another aspect of environment protection. Two methods from mushroom cultivation might serve here as example.

F i r s t , the oyster mushroom, Pleurotus ostreatus, can be grown on straw as sole substrate. It has been found in this country that spent Pleurotus compost, being very nutritious and having a good smell , can be used as an addition to the fodder for cattle. In this way straw, an almost costless agricultural surplus product is total ly recycled, part ial ly giving rise to fruitbodies (ca 10% of substrate weight), part ial ly replacing peat i n gardening, and part ia l ly being used as fodder.

Secondly, experiments in Germany have shown that edible mushrooms can be successfully grown on composted household garbage. The reason, however, why this method has not been introduced into practise is, that unfortunately the average content of heavy metals i n ordinary household is too high and results i n too high contents of these substances i n the fruitbodies produced on this substrate.

F U N G I IN B I O T E C H N O L O G Y IN T H E F U T U R E

In the future many of the biotechnological uses of fungi are expected to continue, even at an increased scale, and new application w i l l be added. Some of these products and areas are listed i n table 6.

Tab. 6 Fungi in biotechnology in the future

Food

Metabolic products Genetic engineering Microbial leaching Biological control of pests and pathogens Environments protection Research

Basic research Space biology

Food Besides using other fungi for the production of new types of food such as

Q U O R N , constantly new strains and species of edible fungi are investigated for mushroom cultivation. A n interesting example is the cult ivation of the edible morel, Morchella conica, based on an U S patent, which is reported to enter now an economically feasible stage (Coombs 1994).

Metabolic products Constantly new, better and more metabolites w i l l be produced i n the future by

fungal species, some of them modified by genetic engineering. This is true for work on the production of " H i r u d i n " ( H O E C H S T A G , Germany) , for human "Insul in" ( N O V O , Denmark) and for new types of brewing yeasts. In a l l cases yeasts are used

because of the relative ease and efficiency of their genetic manipulation, cultivation and metabolic production.

Genetic engineering Genetic engineering usually includes i n principle the following steps. F irs t :

Isolation of the genetic information for the production of an important metabolite from a naturally producing organism (often a higher plant or an animal, usually slow growing and producing). Second: Transfer of that genetic information into another organism (usually a fast growing and fast producing microorganism). T h i r d : Integration of this information into its genetic material. F ina l ly these microorganisms, in our case, fungi, often yeasts, produce w i th modern fermentation technology the desired metabolites faster, in higher quantities and at much lower costs because of their simple growth requirements and modern fermentation technology.

M i c r o b i a l leaching Bacterial leaching of ores by now is a well established method to obtain econom­

ically valuable metals from ores too low in metal content for conventional mining. The principle is that the metal-containing ores are percolated wi th microorganism-containing liquids, the metals are solubilized by the bacterial metabolism and may be later extracted or precipitated. Recently, methods are being developed to employ also fungi (Saccharomyces, Rhizopus), for specific extraction of such valuable metals as manganese, uranium, gold and plat inum (Groger et al . 1987).

Biological control A number of promising projects involving fungi are being investigated, e.g.

control of the deadly chestnut blight or the Dutch elm disease, using hypovirulent strains of the respective fungal pathogens (Adams 1988).

Environment protection A number of projects are concerned wi th the use of fungi for degradation of waste

or toxic materials. Of particular interest are here the often highly toxic xenobiotics, such as polycyclic aromatic hydrocarbons, where research at the Czechoslovak Academy of Sciences and i n our own laboratory has shown that fungi, particularly ligninolytic fungi, are able to degrade a high proportion of these substances in a relatively short time (Vyas et al . 1994a, Vyas et al . 1994b).

Other problems aire caused by our indispensable plastic materials, because they are usually produced from the rapidly diminishing fossil resources and because of their lacking biological degradability (originally considered to be an advantage). As a possible solution new microbial biosynthetic thermoplasts from renewable resources (e.g. B I O P O L , ICI , England) have been developed. However, in order to use them generally and in large quantities, their complete bio-degradability (without toxic residues) has to be established first. Research concerning the abi l i ty

of fungi to degrade these materials is being conducted in our laboratory and the results look quite promising.

Research Also i n the future fungi w i l l be involved i n research. A m o n g these projects

are the elucidation of basic principles like the effect of gravity on l iv ing systems. Because of their low level of organisation and good handability, fungi again are investigated i n the laboratory to solve these questions and because of new and interesting approaches a new aspects of the use of fungi i n space biology research are presented.

The recent developments i n space flight and the possibility to conduct research i n orbit, recently provided the chance to investigate l iv ing systems under space conditions such as (near) weightlessness (microgravity), cosmic irradiation and low pressure. A few examples for the involvement of fungi i n this type of research shall be presented, which i n some cases may lead to new biotechnological processes (Molitoris 1990b).

Genetic engineering often involves optimisation of hybr id formation. Electro-fusion is such a method, where cells are brought into contact i n an electric field, form chains and are hybridised by electric pulses. Experiments using yeast cells under simulated and actual space conditions ( U S / G e r m a n space mission D - l , 1985) have shown a much higher yield of hybrid cells than on the ground since thermoconvection and sedimentation do not interfere.

F ina l l y let us came back to a product where fungi were essential i n biotechnology already in the past and s t i l l are important in the present and certainly w i l l be also in the future, let us speak about beer: In the last U S / G e r m a n spacemission (D-2, 1994), space experiments wi th brewers yeast were conducted. Whether they were successful, the forthcoming publications w i l l show.

Summarising the role of fungi in biotechnology in the past, i n the present and i n the future, we can state:

Fungi i n biotechnology are important and their future certainly looks bright, but it depends on us, on the scientists and the people who use our results, whether our positive expectations may be fulfilled, so that modern biotechnology w i t h fungi does not destroy but protects and improves the blue planet earth on which we live.

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